Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. We have recently identified dynamin 2 (Dyn2), a key regulator of clathrin-mediated trafficking, as a novel Nef binding partner that is required for its ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by RNAi potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells this function of Nef could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The infectivity enhancement by Nef also depended on clathrin, since it was diminished in clathrin- depleted cells and profoundly inhibited in cells expressing the clathrin-binding domain of AP180, which blocks clathrin-coated pit formation. This application focuses on the role of the Nef-Dyn2 interaction in the enhancement of HIV-1 infectivity and replication, because this function of Nef remains poorly understood. One goal is to elucidate what enables Nef to interact with Dyn2 but not with closely related isoforms. In particular, since the two Dyn2 domains which confer Nef binding mediate Dyn2 assembly, we propose to determine whether Nef binding and Dyn2 assembly are linked. Another goal is to understand the relevance of Dyn2 and its interaction with Nef for HIV-1 infectivity and replication. Our preliminary results also indicate that Nef affects the neutralization sensitivity of HIV-1 virions, which could have important implications for HIV-1 replication in vivo. We therefore propose to examine the relationship between the effects of Nef on HIV-1 infectivity and neutralization, the breadth of the effect of Nef on neutralization, and whether Nef affects antibody binding to the Env trimer. PUBLIC HEALTH RELEVANCE The Nef protein of HIV-1 is crucial for the development of AIDS in infected individuals. Nef directly enhances the infectivity of viral particles by an unknown mechanism, and we have recently shown that a human protein called dynamin 2 binds to Nef and is required for its ability to enhance virus infectivity. Since this activity of Nef is likely to affect the ability of HIV-1 to spread in people, we propose to examine what enables Nef to bind dynamin 2 but not closely related proteins, and to investigate the relevance of this interaction for the ability of HIV-1 to infect human cells and to cause disease.